This research identifies several important cell physiological questions that can be uniquely addressed with the help of potentiometric fluorescent indicators and multidimensional microscope imaging. The research is both driven by and drives the application of these methods. Improved potentiometric indicators will be developed not only to serve the needs of this laboratory; we will continue to engage in collaborations for the design of customized indicators for other laboratories and to make these freely available to cell biologists and neuroscientists. The first project describes studies designed to determine mechanisms for intracellular regulation of mitochondrial activity. The laboratory has a unique capability to measure mitochondrial membrane potential quantitatively for individual mitochondria in living cells with a Nernstian fluorescent dye. This provides us the opportunity to combine 2D and 3D quantitative fluorescence imaging to study the interplay of cytosolic calcium, pH, and ATP/ADP ratios, in regulating the components of the mitochondrial electrochemical gradient. In addition, we will develop a method for measuring the other component of the mitochondrial electrochemical potential, the delta-pH. A second project examines stimulus-induced electrophysiological changes in the endoplasmic reticulum membrane of living cells. This organelle is the central cellular store for calcium and is intimately involved in intracellular vesicular traffic. Yet, because of its convoluted structure and its small lumenal volume, it has not been directly studied by electrophysiological or fluorescent indicator techniques. New potential-sensitive ratiometric dyes will be engineered to be soluble in soybean oil; they will then be microinjected into cells for specific labelling of er. The response of the er membrane to cellular stimuli which induce calcium cycling will then be studied via high- resolution dual-wavelength microscope imaging. A third project explores the biological significance of our recent finding that dual wavelength ratiometric potential-sensitive dyes reveal differences in the electrical state of the membrane in different regions along the surface of a neuronal cell. Experiments are proposed to use patch clamp studies to explore the electrophysiological consequences of these lateral differences. We will also use newly synthesized dyes to define the components of the intramembrane potential profile responsible for this intriguing discovery.